CYTOLOGICAL OBSERVATIONS OF THE EFFECT OF ENZYMES ON ACID-TREATED SPORES OF BACILLUS CEREUS

Resting spores of Bacillus cereus were studied cytologically by means of a technique involving both acid and enzyme hydrolysis which was supplemented by selective staining and observation by both bright field and phase contrast microscopy. Enzymes were used singly and in two-enzyme sequences following acid hydrolysis. The results show the artifactitious peripheral structure to be digested by the proteases pepsin and trypsin, and the central structure to be relatively resistant to such action. The nucleases ribonuclease and desoxyribonuclease caused no dissolution of structures, but effected to different degrees a loss of basophilia in the peripheral structure. The former's lesser action led to the conclusion that some ribonucleic acid was associated with the spore's nuclear chromatin in a relatively acid resistant complex, thus, aligning the spore's nucleus with that of other plant and animal tissues in regard to the presence of a ribodesoxyribonucleoprotein complex. Other than effecting the phase contrast phenomenon of reversal of contrast, two-enzyme sequences failed to result in striking increases in degradation and dissolution of the central and the peripheral structures. Buffer and diluent effects on a substrate's subsequent susceptibility to enzyme action and to staining reactions were noted and were in accord with the finding-of other investigators. Osmium tetroxide fixation appeared to increase the subt strate's susceptibility to trypsin action. Magnesium sulfate was shown to inhibis pepsin activity. Potassium permanganate was shown to be an effective mordant for increasing the basophilia of the central structure.